1. Field of the Invention
This invention relates to anticancer agents and the processes for their manufacture.
Cancer is an acute or chronic disease of man which is characterized by abnormal tissue growth and destruction that can be effectively treated with anticancer drugs such as methotrexate.
In Biochemical and Biophysical Research Communications 52:27 (1973), Baugh, Krumdieck and Nair reported on the metabolism of the well known anticancer drug methotrexate to its poly-.tau.-glutamates in human tissues. Nair and Baugh in Biochemistry 12:3923 (1973) reported the chemical synthesis of the poly-.tau.-glutamyl metabolites of methotrexate, the formation of these metabolites in rodent tissues, and the hydrolytic susceptibility of these metabolites to the enzyme "conjugase," derived from hog kidney and human plasma. Methotrexate polyglutamates are relevant to cancer chemotherapy because their formation is related to toxicity; they efflux from the cell at a slower rate than methotrexate and they are more inhibitory to thymidylate synthase and AICAR transformylase.
Continuing work with other antifolates led to the discovery that, like methotrexate, many antifolates undergo polyglutamylation. In 1988, Nair, Nanavati, Gaumont and Kisliuk reported in the Journal of Medicinal Chemistry that, like methotrexate, 10-deazaaminopterin and its 10-ethyl derivative undergo polyglutamylation (J. Med. Chem. 31:181, 1988) and these polyglutamyl derivatives inhibit the enzyme thymidylate synthase more effectively than the parent compound. The potent antileukemic agent N.sup.10 -propargyl-5,8-dideazafolic acid (CB 3717, PDDF) is converted to its polyglutamyl derivatives in normal murine tissues (M. G. Nair, Mehtha and I. G. Nair, Fed. Proc. 45:821, 1986). Nair, Nanavati, Kisliuk, Gaumont, Hsio and Kalman reported in the Journal of Medicinal Chemistry (29:1754, 1986) that the polyglutamyl derivatives of PDDF are more effective in inhibiting thymidylate synthetase.
Cheng, Dutschman, Starnes, Fisher, Nanavati and Nair in Cancer Research (34:598, 1985) and Ueda, Dutschman, Nair, DeGraw, Sirotnak and Cheng in Molecular Pharmacology (30:149, 1986) reported that the polyglutamyl derivatives of PDDF and 10-deazaaminopterin were more inhibitory to human thymidylate synthase than the non-polyglutamylated parent compounds. The antipurine effect of the well-known anticancer and anti-arthritic drug methotrexate has been attributed to its polyglutamyl derivatives inhibiting the enzyme AICAR transformylase (Allegra, Drake, Jolivet and Chabner, Proc. Nat. Acad. Sci. USA 82:4881, 1985). These data taken together clearly show that the toxicity of classical antifolates like methotrexate, 10-deazaaminopterins and PDDF is potentiated by polyglutamylation. The accumulation of their toxic metabolites in normal human tissues such as kidneys, bone marrow and liver undoubtedly contributes to the undesirable side effects of these drugs and seriously undermines their clinical utility.
Therefore, it was of interest to us to develop classical antifolates that are powerful inhibitors of the target enzyme dihydrofolate reductase, but incapable of elaboration in vivo to their polyglutamyl derivatives. If these compounds are transported as efficiently as methotrexate to tumor cells, then such compounds should have clinical utility as anticancer drugs and exhibit lower host toxicity. Compounds 1 and 2 have shown excellent antitumor activity in three tumor models (Table I). Both compounds were similar to methotrexate in their efficacy in inhibiting the target enzyme dihydrofolate reductase (Table II). As shown in Table III, compound 1 is transported more efficiently to H35 hepatoma cells than methotrexate as determined by its ability to compete with folinic acid transport in this cell line.
Compounds 1 and 2 were evaluated as substrates and inhibitors of purified human leukemia cell folypolyglutamate synthetase (FPGS). It has been established that substrates of FPGS are capable of polyglutamylation in vivo and the relative magnitude of substrate activity of an antifolate to this enzyme compared to a standard is a measure of its relative ability to undergo polyglutamylation in vivo. In table IV, the relative substrate activities of compounds 1 and 2 are presented compared to two standards, aminopterin and 10-deazaaminopterin. The data establish that the new compounds 1 and 2 are not substrates of CCRF-CEM human leukemia cell folylpolyglutamate synthetase.
Therefore, by analogy to methotrexate, compounds 1 and 2 should have clinical utility as novel anticancer drugs capable of exhibiting lower host toxicity. In addition, they should be useful in the treatment of rheumatoid arthritis since they are expected to be immune suppressants to a similar degree as methotrexate.
This invention accordingly also provides a process for treating leukemia, ascitic and solid tumors and rheumatoid arthritis which comprises administering to a warm-blooded animal with an abnormal proportion of leukocytes or other evidence of malignancy or rheumatoid arthritis a therapeutic nontoxic amount of compounds 1 and 2 as such, or in the form of a pharmacologically acceptable salt thereof.
The process of the invention for the preparation of 4-amino-4-deoxy-10-methylpteroyl-.tau.-methyleneglutamic acid (1) is a synthesis in which commercially available 4-amino-4-deoxy-10-methylpteroic acid (3) is coupled with diethyl-.tau.-methylene glutamate (5) followed by base hydrolysis (Scheme 1). Likewise, the process of the invention for the preparation of 4-amino-4-deoxy-10-methylpteroyl-3-hydroxyglutamic acid (2) is a synthesis in which 4-amino-4-deoxy-10-methylpteroic acid is coupled with dimethyl-3-hydroxyglutamate followed by base hydrolysis (Scheme 2).
Stage I (Scheme 1) is essentially the conversion of 4-amino-4-deoxy-10-methylpteroic acid (3) to the corresponding mixed anhydride (4) by treatment with an equal amount of alkylchloroformate such as isobutylchloroformate in an appropriate solvent such as dimethylsulfoxide (DMSO) in the presence of an acid acceptor, preferably a tertiary amine such as triethylamine or N-methylmorpholine. This reaction can be carried out within a temperature range of 0.degree.-30.degree. C. Other acid acceptors such as substituted pyridines, tributylamine, collidine, lutidine or MgO may be substituted for triethylamine. The reaction may be conducted with other alkylchloroformates such as methyl, ethyl, propyl, etc. Other solvents such as dimethylformamide, hexamethylphosphoramide or dimethylacetamide may be used for this reaction.
In stage II the mixed anhydride 4 is reacted with an excess amount of a diester of .tau.-methyleneglutamic acid such as diethyl-.tau.-methyleneglutamic acid (5). Dimethyl, dibenzyl or di-t-butyl-.tau.-methylene glutamic acid may be substituted for the diethyl derivative. Diethyl-.tau.-methylene glutamate may be added as the hydrochloride form to the mixed anhydride solution 4 followed by the addition of an equal amount or an excess of an acid acceptor such as triethylamine. Alternatively, diethyl-.tau.-methyleneglutamate hydrochloride may be dissolved in a suitable solvent such as DMF, neutralized with an equivalent amount of the acid acceptor, and then added to the mixed anhydride solution 4. This reaction mixture is stirred for 18 hours at 25.degree.-30.degree. C. and the solvent is removed by evaporation under reduced pressure. The resulting coupled product is stirred with an excess of a mixture of 0.1N NaOH and acetonitrile for 18 hours to give the target compound 1. The glutamic acid derivative 1 is soluble in alkali and can be isolated as a precipitate by the addition of an acid to the basic hydrolysate. The precipitate can be recovered by filtration, washed and dried.
For the preparation of compound 2 (Scheme 2), the solution of the mixed anhydride 4 is reacted with an excess amount of the diester of 3-hydroxyglutamic acid such as dimethyl-3-hydroxyglutamate (6). Diethyl, dibenzyl or di-t-butyl-3-hydroxyglutamate may be substituted for the dimethyl derivative. Dimethyl-3-hydroxyglutamate (6) may be added to the solution of the mixed anhydride as the hydrochloride followed by the addition of an equivalent or excess amount of the acid acceptor such as triethylamine. Alternatively, dimethyl-3-hydroxyglutamate hydrochloride may be dissolved in a suitable solvent such as DMF, neutralized with an equivalent amount of the acid acceptor such as triethylamine or N-methylmorpholine and then added to the mixed anhydride solution. The reaction mixture is then stirred for 18-24 hours at 25.degree.-30.degree. C. and the solvent is removed by rotary evaporation under reduced pressure. The resulting product is stirred with an excess of a 3:1 mixture of 0.1N NaOH:acetonitrile for 10-18 hours to give target compound 2. Compound 2 is soluble in alkali and it can be isolated as a precipitate by addition of an acid such as glacial acetic acid to the hydrolysate. The precipitate can be recovered by filtration, washed and dried.